Damage-related changes in the cerebellum of juvenile Oncorhynchus masou: reactivation of neurogenic niches and astrocytic response

In the cerebellum of juvenile Oncorhynchus masou, proliferating BrdU+ and HuCD+ cells and constitutive neurogenic niches were detected in different zones; the largest number of labeled cells were found in the dorsal part of the molecular layer and the dorsal matrix zone (DMZ). Cells labeled with glutamine synthetase (GS) and radial glia were also present in the intact O. masou cerebellum. The most intensive proliferation was detected in the rostral part of cerebellum. This part is assumed to contain active zones of constitutive neurogenesis. After an injury inflicted to the cerebellum, the number of BrdU+ and HuCD+ cells increased significantly. The number of BrdU+ cells after this type of injury was much greater than after a telencephalon trauma. A quantitative analysis revealed that after the cerebellum injury the proliferative activity in the caudal part of CCb is increased compared to that in the control. A reactivation of neurogenic and neuroepithelial niches and their transformation into reactive neurogenic domains, with an increased distribution density of intensely labeled HuCD+ cells of different types, were observed. The increase in the number of HuCD+ differentiated cells in the basal area suggests that the processes of neuronal differentiation are intensified in the cerebellum of juvenile O. masou after injury. The number of GS positive cells (GS+) and fibers increased in all the zones of cerebellum. The most intensive astrocytic response was noted in the dorsal part of cerebellum. The data of the enzyme immunoassay confirm the multiple variations in the level of GS after a traumatic injury to cerebellum in O. masou.


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Fish brain, growing with the body throughout the life, is an interesting model for studying the 35 brain recovery after a traumatic injury and the influence of various factors on these processes. In  Various types of damage to fish brain create special conditions for the implementation of genetic 44 programs that enhance the proliferation of progenitor cells, and also activate the neurogenic ELISA immunoassay of the quantitative level of GS in the brain of intact fish and fish after the 182 cerebellum injury was performed using a Fish Glutamine Synthetase ELISA Kit (MBS011386, 183 USA). For the enzyme immunoassay, we used the material from intact animals and fish at 1h, 184 3h, 12h, 1d, 2d, 3d, and 5 days after the mechanical injury of cerebellum (n = 3 in each group). 185 The brain of intact and experimental fish was removed from the skull in a 0.02M phosphate 186 buffer, weighed and then thoroughly washed in an ice-cooled 0.02 M phosphate buffer (pH 7.2) 187 to remove blood. The brain tissue was further mechanically cut into small pieces in 5 mL of 188 phosphate buffer in a Potter-Elvehim PTFE glass homogenizer (Sigma, Aldrich, USA) on ice. 189 The O. masou brain homogenates contained 10 mg tissue per 100 μL of PBS. The resulting 190 suspension was sonicated on a Sonoplus 2070 ultrasonic homogenizer (Bandelin,Germany,191 Berline) to destroy cell membranes. The homogenates were then centrifuged for 15 minutes at   To characterize cerebellum, the dorsal, lateral, and basal regions of corpus cerebellum (CCb) 244 were examined ( Figure 1A). The cell composition of all the cerebellar regions included five 245 morphological types of cells, the morphometric parameters of which are given in Table 1.    In the ganglionic layer, the soma of pear-shaped Purkinje cells (type III) and elongated bipolar 276 eurydendroid cells (EDC) (type IV) was stained with toluidine blue ( Table 1). 277 In the surface part of the molecular layer of the lateral zone, the tangential migration of a large 278 number of type II (rod-shaped) cells was revealed after injury ( Figure 1C). The patterns with 279 tangential cell migration were not detected in the control animals. In the dorsal zone near  In the granular layer of cerebellum, most of type V granular cells were stained with toluidine 285 blue; the size of their soma was ca.5 μm (Table 1). In the dorsal and lateral zones of the granular 286 layer, the number of cells decreased after the injury (Figure 2A and D). In the basal zone, the 287 number of cells somewhat increased ( Figure 2G). In the granular layer of the cerebellum,   the GrLs averaged at 20 ± 2 cells (P < 0.05, Figure 2H). In the MPML, the number of labeled 317 cells decreased twofold. (Figure 2H). In the MPML of the dorsal and basal zones, the intensely 318 BrdU-labeled cells had a low density of distribution. In aggregations of BrdU+ cells, the 319 intensity of labeling was sometimes lower than that of individual cells.    as low as that in the rostral part of cerebellum ( Figure 2B). In the GrL of the caudal part, the number of BrdU+ cells was significantly lower than in the rostral one (P < 0.05, Figure 2B and 360 H). In different areas of the molecular layer, no significant differences in the number of labeled 361 cells were found between the rostral and caudal parts of cerebellum.

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In the lateral zone of the caudal part, there were separate labeled cells and small groups of cells 363 in the MPML. The number of BrdU+ cells in the SPML was 1.7 times as low as that in the 364 rostral part (Figure 2E). In the basal zone, no significant differences in the number of labeled 365 cells in the molecular layer between the rostral and caudal parts were observed ( Figure 2H). 366 However, in the ventral apex of the basal zone, the number of BrdU+ cells increased distinctly 367 and amounted to 45 ± 7 in the DPML ( Figure 3C). In the rostral part of the corresponding zone,

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there were no apparent clusters of cells ( Figure 2H).

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The results of the quantitative analysis showed the maximal proliferative index in the caudal part    Student's t-test was used to determine significant differences between the control animals and 402 those after injury (n = 5 in each group; *P < 0.05 vs. control group).

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In the rostral part, the number of labeled cells in the molecular layer in the lateral zone increased 405 after injury, especially in the SPML (Figure 4C), and in the caudal one it increased ( Figure 4D). 406 The most significant increase in the number of BrdU+ cells was detected in the GrL (   injuries also showed that the total number of proliferating cells after the cerebellum injury is 478 higher than that after the telencephalon one (P < 0.05, Figure 7A    was similar to those in the body of the cerebellum: 12.1 ± 1.8% in the intact cerebellum and 21.1 510 ± 2.7% after injury ( Figure 1F). 511 After the telencephalon injury, the number of BrdU+ cells was 2.6 times as low as that in the 512 intact cerebellum and 4.5 times as low as that in the injured cerebellum. Intensely labeled BrdU+ 513 cells were located at a small distance from each other at the surface of granular eminences, in 514 most cases forming intensely labeled clusters.  Table 1. Type VI 520 cells had the rounded soma, but their size was significantly smaller ( Table 1). Most of them 521 were located singly or, in some cases, grouped into two elements in the MPML or, sometimes, 522 near the end of GS+ fibers. In the molecular layer, the distribution density of GS+ elements was 523 higher than that in the granular layer. analysis, 5 ± 2 fragments of fibers per view field were found in the control animals ( Figure 9A).

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Type VII cells in the intact animals were located in the molecular layer, infraganglionic plexus,   Table   579 1. 580 After the injury, the number of intensively labeled GS+ cells of type VII in the dorsal, lateral, 581 and basal zones increased 1.9-fold as compared with the control (Figure 9A, C, E and G). Type 582 VI cells with moderate GS activity were located singly, but more intensely labeled type VI cells  injury. Thus, in the control animals, the GS concentration was 15.7 ± 2.6 pg/mL; at 1 hour post-injury, on average 21.8 ± 1.8 pg/mL (Figure 10). During the first 24 hours post-injury, the 613 maximum GS concentration was recorded at 3 h, 23.4 ± 1.9 pg/mL, and reached the control level 614 at 12 h, 15.8 ± 3.6 pg/mL (Figure 10). Further observations showed that at 1 day after the 615 cerebellum injury, the concentration of GS was 18.5 ± 2.3 pg/mL; at 2 day, it increased to 22.9 ± 616 0.55 pg/mL (Figure 10). On day 3 post-injury, the GS level again decreased to 16 ± 2.2 pg/mL 617 and reached a maximum level of 24.2 ± 0.6 pg/mL by day 5 (Figure 10). Thus, the ELISA data   Table 1). 632 In the intact cerebellum, the largest number of HuCD+ cells was detected in the dorsal In the intact cerebellum, the pear-shaped Purkinje cells were detected in the ganglionic layer 664 ( Table 1). The greatest number of such cells was found in the basal zone ( Figure 11C). Pear-665 shaped cells were labeled in the proximal areas of dendrites. Intensely labeled EDC were found 666 in the dorsal, lateral, and basal zones ( Table 1). Their number was significantly lower than in 667 other cell forms: 6.5% in the dorsal, 7.5% in the lateral, 12.3% and in the basal zones.  (Figure 12A, B and C).
In the basal area, intensely labeled undifferentiated type 1 cells were detected in some cases, but 690 they were not found in the other zones. In the basal area, the greatest number of rounded type 691 VIII cells was recorded after the injury (Figure 12C and 11F). Large type IV cells were detected 692 in the deep layers of all zones of the cerebellum (Figure 11E).The number of intensely labeled, 693 rounded type VIII cells, sometimes with processes, was significantly larger after the injury. The 694 largest number of type IV and V cells was detected in the basal zone ( Figure 11E).

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It has been established that after a brain injury in fish the rate of cell proliferation is much higher 807 than in the absence of damaging effects. According to Clint and Zupanc (2001), the density of

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Concerning the synaptogenesis of the granular layer, it appears that, as in other vertebrates, the 949 mossy fibers, the granule cells, and the Golgi cells participate in the formation of glomeruli, 950 which are the synaptic complexes of this layer (Pouwels, 1978c). Mossy fibers contain more 951 neurofilaments than other axons in the cerebellum of trout and can easily be recognized. In trout, 952 17-mm mossy fiber synapses are observed for the first time. In this stage, the rosette has a rather 953 simple form (Pouwels, 1978c) and contacts with only a few dendrites of granule cells. These  We suggest that metabolic glutamate, which is presumably involved in the morphogenetic increases; however, we do not rule out that these changes are not generalized, but are a particular 975 display of the change in GS activity on day 3 post-injury. This hypothesis is also confirmed by 976 the data of the enzyme immunoassay (ELISA), carried out by us on cerebellum of juvenile O. 977 masou. Thus, in the course of long-term monitoring, a gradual increase in GS activity was 978 observed on day 2 and its slight decrease on day 3 after the cerebellar damage (Figure 10). 979 Nevertheless, these changes in the metabolic activity of GS were only a local decrease in the 980 activity of the enzyme, displayed on day 3. As a result of ELISA, it was found that the activity of indicates a sufficiently high level of production of the enzyme, whose activity can decrease 991 cyclically and be determined by various factors, the nature of which is still unknown.

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The increase in the number of GS+ cells in the molecular and granular layers and the increase in 993 the activity of the enzyme on day 3, observed by us, can be attributed to the astrocyte response 994 observed during the damage. However, the concentration of such cells is not high enough to 995 relate this response of GS+ cells to the formation of an astrocytic response like that after an 996 injury in mammalian CNS. As is known, a pool of reactive astrocytes, with their morphological 997 and biochemical features being significantly different from normal astrocytes, is formed after a traumatic impact on brain of mammals (Revett et al., 2013). The cellular mechanisms associated 999 with the transformation of astrocytes population and the allocation of a subpopulation of 1000 activated astroglia in brain of fish have not been studied yet. In contrast to astrocytes of determine significant differences in regenerative abilities between mammals and fish (Zupanc